Self-oligomerization is essential for enhanced immunological activities of soluble recombinant calreticulin

• Published in: PloS one 2013-06, Vol.8 (6), p.e64951-e64951
• Main Authors: Huang, Shang-Hui, Zhao, Li-Xiang, Hong, Chao, Duo, Cui-Cui, Guo, Bing-Nan, Zhang, Li-Juan, Gong, Zheng, Xiong, Si-Dong, Gong, Fang-Yuan, Gao, Xiao-Ming
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Arthritis; B cells; Binding sites; Biochemistry; Biology; Blotting, Western; Calreticulin; Calreticulin - chemistry; Calreticulin - immunology; Calreticulin - metabolism; Chromatography, Affinity; Dimerization; E coli; Endocytosis; Endoplasmic reticulum; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Gel filtration; Heat shock proteins; Hypotheses; Immunology; Lectins; Lipopolysaccharides - pharmacology; Liver - metabolism; Macrophages; Macrophages, Peritoneal - drug effects; Macrophages, Peritoneal - immunology; Macrophages, Peritoneal - metabolism; Mice; Mice, Inbred C57BL; Microenvironments; Monomers; Oligomerization; Oligomers; Polypeptides; Protein Multimerization - immunology; Recombinant Proteins - chemistry; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; Residues; Rheumatoid arthritis; Rheumatoid factor
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0064951

We have recently reported that calreticulin (CRT), a luminal resident protein, can be found in the sera of patients with rheumatoid arthritis and also that recombinant CRT (rCRT) exhibits extraordinarily strong immunological activities. We herein further demonstrate that rCRT fragments 18-412 (rCRT/18-412), rCRT/39-272, rCRT/120-308 and rCRT/120-250 can self-oligomerize in solution and are 50-100 fold more potent than native CRT (nCRT, isolated from mouse livers) in activating macrophages in vitro. We narrowed down the active site of CRT to residues 150-230, the activity of which also depends on dimerization. By contrast, rCRT/18-197 is almost completely inactive. When rCRT/18-412 is fractionated into oligomers and monomers by gel filtration, the oligomers maintain most of their immunological activities in terms of activating macrophages in vitro and inducing specific antibodies in vivo, while the monomers were much less active by comparison. Additionally, rCRT/18-412 oligomers are much better than monomers in binding to, and uptake by, macrophages. Inhibition of macrophage endocytosis partially blocks the stimulatory effect of rCRT/18-412. We conclude that the immunologically active site of CRT maps between residues 198-230 and that soluble CRT could acquire potent immuno-pathological activities in microenvironments favoring its oligomerization.